The enzyme thymidylate synthase provides a unique pathway for de novo biosynthesis of dTMP in mammalian cells, and is considered an important target in mediating the cytotoxicity of 5-fluorouracil (FUra) in human colon carcinomas. This application concerns three aspects of thymidylate synthase as a target for therapy in colon carcinoma. These are a) modulation of the interaction of 5-fluorodeoxyuridylate (FdUMP) with thymidylate synthase, b) the role of dThd salvage in intrinsic resistance, and hence the significance of thymidylate synthase as a target for tumors in situ, and c) the consequences of FUra base substitution in thymidylate synthase mRNA. Modulation studies will examine the consequences of the addition of polyglutamate residues to N5, N10-methylenetetrahydrofolate (CH2-H4PteGlu-n), in terms of affinity for thymidylate synthase purified from human colon adenocarcinoma xenografts, and effect on the kinetics of the catalytic reaction, the binding of FdUMP, and stability of covalent ternary complexes. The distribution of CH2-H4PteGlu-n in colon xenografts and the effect of leucovorin administration will be examined. The role of dThd salvage in determining the sensitivity of xenografts to FUra will be examined. Specifically, cloned colon adenocarcinoma cell lines deficient in thymidine kinase activity (TK-) will be selected in tissue culture and characterized by TK activity, clonogenic survival after FUra, and drug metabolism. In vivo models will be developed by growing clonal parent and TK- variants in immune-deprived mice with characterization as above. In a cell free system, the effect of FUra substitution at specific frequencies into thymidylate synthase mRNA will be examined with respect to alteration of the rate and function of in vitro synthesized mRNA, and the rate of translation and function of translated protein. The long term objectives are to a) identify potential targets for chemotherapeutic agents using cell free systems, and b) determine their significance in the treatment of human colon cancer by further development of relevant in vitro and in vivo models of human colon adenocarcinoma in which treatment strategies and hypotheses may be tested.